L7: Cell Culture

Question 1

What are advantages of cell cultures?

Answer 1

→Control of the physiochemical environment (pH, temperature, osmolarity) and physiological conditions (levels of hormones and nutrients)

→Control of the micro-environment of the cells

→Cells can be easily characterised by cytological or immune-staining techniques and visualised using imaging techniques

→Cells can be stored in liquid nitrogen for long periods (cryopreservation)

→Cells can be easily quantified
Reduces use of animals in scientific experiments

→Cheaper to maintain

Question 2

What are the two types of cell cultures?

Answer 2

→primary tissue cells

→immortalised cell lines

Question 3

What are the differences between primary tissue cells and cell line culture?

Answer 3

→primary tissue cells have limited lifespan retain cells
but CL has infinite lifespan and loses specificity
→pre-characterised and ready to use but CL requires authentication required before use
→study cells with varied donor characteristics but CL study single donor repeatedly
→PTC are less heterogenous

Question 4

What are the characteristics of PTC?

Answer 4

→Cells derived directly from tissues/patients (unmodified)
→Finite lifespan (~6-7 divisions)

→Cells divide and/or differentiate

→Cells carry out normal functions

→Living conditions similar to body environment

Question 5

What are the two methods for isolation of cells?

Answer 5

→explant culture-Cells allowed to migrate out of an explant
→Mechanical (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAase)

Question 6

Why are haemotopoeic cells an exception when isolating cells?

Answer 6

→Do not need to be disaggregated
→already are as individual cells circulating in blood.
→Use density centrifugation instead

Question 7

Why is centrifugation used for haematopoietic cells?

Answer 7

→Takes advantage of the different densities of diff blood cells

Question 8

Which cells are densest?

Answer 8

→Granulocytes

Question 9

What are other methods of isolation?

Answer 9

→Immuno-purification- using antibody-coated magnetic beads

→Fluorescence activated cell sorter

Question 10

What are the non-haematopoietic cells?

Answer 10

→liver
→muscle
→endothelial
→skin
→nerves
→ fibroblasts

Question 11

What are the disadvantages of primary tissue cells?

Answer 11

→Inter-patient variation
→Limited number (small amount at high cost)
→Finite lifespan and hard to maintain
→Difficult molecular manipulation
→Phenotypic instability
→Variable contamination

Question 12

What are the characteristics of cell lines?

Answer 12

→Immortalised cells from one type of primary tissue cells

→Less limited number of cell divisions (~30) or unlimited

→Phenotypically stable, defined population

→Limitless availability
→Easy to grow
Good reproducibility

→Good model for basic science if same cell lines are used

Question 13

What are the two types cell lines?

Answer 13

1. Isolated from cancerous tissues (e.g. HeLa cells)

2. Immortalisation of healthy primary cultures (usually through genetic manipulation)

Question 14

What are the targets in cells for production of cell lines through genetic manipulation?

Answer 14

→processes that regulate cellular growth and ageing

Question 15

What are process that regulate cellular growth and ageing?

Answer 15

→p53
→pRB
→telomerase

Question 16

What happens as telomeres shorten?

Answer 16

→cell division stops

→ Apoptosis (p53, pRb)

Question 17

How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?

Answer 17

→taking advantage of viral ‘oncoproteins

Question 18

What are the viruses used to alter a cell’s capability for its finite number of divisions and their viral oncoproteins?

Answer 18

→Simian Virus-40
(SV40)- Large T antigen, Small t antigen
→Human Papilloma
Virus (HPV), E6 E7

Question 19

What are the targets for viruses used in cell line division numbers?

Answer 19

→p53

→pRb

Question 20

How does SV40 T-antigen work in cell line generation?

Answer 20

→interacts with p53 and pRb

Question 21

What does use of SV40 T-antigen in cell line allow?

Answer 21

→increased growth without loss of function of these proteins

Question 22

What does E6 and E7do?

Answer 22

→targets p53 for degradation, and E7 binds to pRb inactivating it

Question 23

What is the phenotype like in cell lines made using E6 and E7?

Answer 23

→maintain a differentiated phenotype

Question 24

What do some cells need in generating cell lines?

Answer 24

→need both introduction of the telomerase gene

→inactivation of the pRb/p53 for “immortalisation”

Question 25

Are TERT enzymes active in somatic cells?

Answer 25

→no

Question 26

How is TERT activated in somatic cells?

Answer 26

→need to be transfected →Make a vector containing selection marker eg neomycin(antibiotic resistance) and telomerase gene. →Plasmid has to be circularised →Treat cells with antibiotic and only cells with it will survive

Question 27

What are the advantages of 2D cell culture?

Answer 27

Simple, well established | - Affordable

Question 28

What are the disadvantages of 2D cell cultures?

Answer 28

→Forced apical-basal polarity →High stiffness →Limited communication with other cells →No diffusion of gradients →Results not relevant to human physiology →Cells lose their original phenotype and functional characteristics

Question 29

What are the advantages of 3D cultures?

Answer 29

→Adhesion in all three dimensions →No forced polarity → Variable stiffness → Diffusion gradients of nutrients and waste products →More relevant to human physiology →Allow cells to aggregate- balls of cells

Question 30

What are the disadvantages of 3D cultures?

Answer 30

→More complex | →Added expense

Question 31

What are the two types of shapes of 3D cultures?

Answer 31

→spheroids | →organoids

Question 32

Describe spheroids

Answer 32

→3D →cellular aggregate composed of 1(or more) cell types that grow and proliferation →exhibit enhanced physiological responses →do not differentiate or self-organise

Question 33

Describe organoids

Answer 33

``` →3D culture →derived from PSCs, AdSC → cells spontaneously self organise into properly differentiated functional cells types → resemble in vivo counterpart →resemble some function of organ ```

Question 34

What do patient derived organoids allow?

Answer 34

→the study cancer drug resistance | →Make treatment based on results of the treatment on culture

Question 35

Why are patient-derived cells organoids?

Answer 35

→derived from primary tissue cells

Question 36

What is the percentage mimicry of phenotype in organoids?

Answer 36

→85%

Question 37

Define transefection

Answer 37

process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab.

Question 38

Give examples of non-viral transfection methods

Answer 38

→plasmid, | → CRISPR/Cas9 complex

Question 39

Give examples of chemical transfection

Answer 39

→Lipofection →Calcium phosphate →Cationic polymer →Magnet-mediated transfection

Question 40

Why is transfection performed in 2D?

Answer 40

→if 3D not all cells would be transfected

Question 41

Give examples of physical transfection

Answer 41

``` →Electroporation →Nucleofection →Microinjection →Laserfection/ optoinjection →Biolistic Particle Delivery ```

Question 42

What is lipofection?

Answer 42

→Using cationic lipid transfection systems | →uses liposomes

Question 43

Why are liposomes used for lipofection?

Answer 43

→has unilamellar liposomal structure →has phospholipid bilayer →net positive charge and the DNA/plasmid will have a negative charge

Question 44

Describe lipofection

Answer 44

``` 1. Interaction with the cell membrane 2. Taken up by endocytosis 3. Release from the endosome →4. Transport to the nucleus →5. Entry to the nucleus inefficient and may need mitosis ```

Question 45

Do liposomes deliver hydrophobic or hydrophilic drugs?

Answer 45

→hydrophobic or hydrophilic

Question 46

How do you ensure the drug is delivered into the tissue of interest?

Answer 46

→Attach tissue specific antigens to the liposomes

Question 47

How is electroporation carried out?

Answer 47

→High electric field →Forms pores- increase permeability →DNA goes through cell which then reseal

Question 48

What does the rate of pore sealing in electroporation dependent on?

Answer 48

→temperature

Question 49

What are the characteristics of nucleofection?

Answer 49

→Combination of electroporation and lipofection - Increased efficiency particularly of non-dividing cells - Technology is protected under patent - Different solution and protocols are used for each cell type

Question 50

Describe viral infection

Answer 50

``` 1. plate HEK293T cells →transfect plasmids →collect 1st supernatant →refrigerate →collect 2nd supernatant →collect viral pellet after centrifuging →use for transduction ```

Question 51

What is HEK293T?

Answer 51

→used as vehicle for production of material in viral transfection

Question 52

What types of cells are used in viral infection?

Answer 52

→Retrovirus, →Adenovirus, →Lentivirus commonly are used.

Question 53

What enables HEK293T cells to produce recombinant proteins within plasmid vectors?

Answer 53

→contains the SV40 large T antigen